Early Life Arsenic Exposure and Acute and Long-term Responses to Influenza A Infection in Mice

Background: Arsenic is a significant global environmental health problem. Exposure to arsenic in early life has been shown to increase the rate of respiratory infections during infancy, reduce childhood lung function, and increase the rates of bronchiectasis in early adulthood. Objective: We aimed to determine if early life exposure to arsenic exacerbates the response to early life influenza infection in mice. Methods: C57BL/6 mice were exposed to arsenic in utero and throughout postnatal life. At 1 week of age, a subgroup of mice were infected with influenza A. We then assessed the acute and long-term effects of arsenic exposure on viral clearance, inflammation, lung structure, and lung function. Results: Early life arsenic exposure reduced the clearance of and exacerbated the inflammatory response to influenza A, and resulted in acute and long-term changes in lung mechanics and airway structure. Conclusions: Increased susceptibility to respiratory infections combined with exaggerated inflammatory responses throughout early life may contribute to the development of bronchiectasis in arsenic-exposed populations. Citation: Ramsey KA, Foong RE, Sly PD, Larcombe AN, Zosky GR. 2013. Early life arsenic exposure and acute and long-term responses to influenza A infection in mice. Environ Health Perspect 121:1187–1193; http://dx.doi.org/10.1289/ehp.1306748


Cytokines
Inflammatory cytokines were measured in BALf supernatants using a mouse inflammation Cytometric Bead Array (BD Biosciences, San Diego, CA, USA) as per the manufacturer's instructions. Measurement of total protein content of the BALf was carried out using the Bradford technique employing the Bio-rad Protein Assay kit according to manufacturer instructions (BIO-RAD, NSW, Australia).

Thoracic gas volume and lung mechanics
Lung volume and lung mechanics were measured in offspring at 7 days, 21 days and 7 weeks post-infection, and in mice exposed to arsenic in adulthood only. To measure lung function in vivo, mice were anaesthetized by intra-peritoneal injection of a mixture containing xylazine (1 mg/mL; Troy Laboratories, New South Wales, Australia) and ketamine (20 mg/mL; Troy Laboratories, New South Wales, Australia) at a dose 0.1 mL/10 g body weight. Mice were tracheotomised and a 10 mm long tracheal cannula inserted (23G stainless steel for 2 and 4 week old mice; 1.26 mm outer diameter polyethylene tube for mice 8 weeks or older) and secured with suture. Mice were ventilated (MiniVent, Harvard Apparatus, Germany) at a tidal volume of 10 mL/kg, respiratory rate of 400 breaths per minute and positive end expiratory pressure of 2 cmH 2 O.
Plethysmography was used to measure thoracic gas volume (TGV) as described previously (Janosi et al. 2006). Briefly, the trachea was occluded at end expiration (transrespiratory pressure, P rs = 0 cmH 2 O) and the intercostal muscles were stimulated with intramuscular electrodes to induce inspiratory efforts. Six 20 V pulses of 2-3 ms in duration were delivered over a 6 s period while recording changes in tracheal pressure and plethysmograph box pressure.
TGV was calculated using Boyle's law after correcting for the impedance and thermal properties of the plethysmograph (Janosi et al. 2006).
Lung mechanics were measured using the forced-oscillation technique as described previously (Sly et al. 2003). The forcing function (9 frequencies from 4 -38 Hz) was generated by a loudspeaker and delivered to the animal via a wave tube during pauses in ventilation. The respiratory system impedance spectrum (Z rs ) was measured and a 4-parameter model with constant phase tissue impedance was fitted to the data to partition Z rs into components representing the mechanical properties of the airways and parenchyma (Hantos et al. 1992). This model allowed the calculation of airway resistance (R aw ) and inertance (I aw ) and coefficients of tissue damping (G) and elastance (H). The resistance and inertance of the tracheal cannula were subtracted from R aw and I aw respectively. As most of the inertance is contained in the tracheal cannula, values of I aw were insignificant and not reported.

Responsiveness to Methacholine
Hyper-responsiveness of the respiratory system to bronchoconstricting agents, such as methacholine, can reflect the presence of pulmonary inflammation or altered lung structure such as excess mucous production or increased airway smooth muscle (Lundblad et al. 2007(Lundblad et al. , 2008. Airway responsiveness to methacholine (MCh) was measured in offspring at 7 weeks postinfection and mice exposed to arsenic as adults only. Methacholine was prepared using Acetylβ -methylcholine chloride (minimum 98%) (Sigma-Aldrich, St Louis, MO, USA) dissolved in sterile saline to concentrations of 0.1, 0.3, 1, 3, 10 and 30 mg/mL. Mice received a saline aerosol followed by increasing doses of aerosolized MCh from 0.1 to 30 mg/mL for 90 seconds. Lung function was measured every minute for 5 minutes after the conclusion of each aerosol. Dose response curves were constructed from the maximal response per dose of MCh.

Airway remodeling
Following euthanasia, the lungs of offspring at 7 weeks post-infection were fixed through intratracheal instillation of 2.5 % gluteraldehyde at 10 cmH 2 0. The left lung was isolated and bisected into superior and inferior portions at the entrance of the left primary bronchus. The inferior portion of the left lung was embedded with the bisected face down to obtain transverse cross sections of the primary bronchus for airway morphology. Airway sections, 5µm thick, were cut at proximal, middle and distal parts of the lung for airway histology. To calculate the area of the airway smooth muscle (ASM) airway sections were stained with Masson's Trichrome (Sigma Aldrich, Castle Hill, NSW). The ASM layer was traced at 100x magnification in circular airways using stereological software (newCAST, Visiopharm, Hørsholm, Denmark) and a motorized microscopic stage. To normalize for airway size, the square root of the area of ASM layer was divided by the perimeter of the basement membrane (P bm ). The histological scoring was performed by the corresponding author who was blind to the treatment group of each sample until final analysis was performed. To calculate the number of mucous producing cells airway sections were stained with Alcian Blue (Sigma Aldrich, Castle Hill, NSW) to measure mucous producing cells. Mucous cell expression was calculated as the percentage of mucous positive cells divided by the total number of epithelial cells in the airway. Airways were classified by their basement membrane perimeter (large >1,500 µm, medium > 1,000 µm, small <1,000 µm) (Hirota et al. 2006).